PROJECT SUMMARY The goal of the proposed program is to design and synthesize new emissive nucleoside and nucleotide analogs and implement them as probes for monitoring nucleoside- and nucleotide-based transformations as well as nucleic acids function, structure, dynamics and recognition. Advancing effective fluorescence-based tools for exploring nucleoside, nucleotide and oligonucleotides, as well as their metabolism, regulatory processes and interactions with potential therapeutic agents will further knowledge and advance new diagnostic approaches, facilitating drug discovery. Specifically, we will address: AIM 1. To design, synthesize and incorporate new isomorphic fluorescent nucleoside and nucleotides. The main design criteria include: (i) High structural similarity to the native nucleobases to faithfully mimic their size and shape, as well as hybridization and recognition properties, (ii) Red shifted absorption spectrum to minimize overlap with the absorption of the natural bases, and (iii) Adequate emission quantum efficiency and visible emission wavelengths. Efficient synthetic and enzymatic pathways will be devised, providing the nucleosides, nucleotides and oligonucleotide. AIM 2. To photophysically and biophysically characterize the modified nucleosides and oligonucleotides. The photophysical characteristics (e.g., absorption and emission maxima, quantum yield and brightness, excited state lifetime, as well as susceptibility to environmental polarity and static and dynamic quenching by native nucleosides) will be rigorously evaluated and interpreted. The outcome of these analyses and the data generated dictate the utility and potential applications of the nucleoside surrogates. AIM 3. To implement the promising emissive analogs in biophysical, biochemical and discovery assays. These assays will facilitate: (i) Studying the enzymatic deamination of adenosine to inosine, which occurs in three key biological contexts: purine metabolism, mRNA editing and tRNA maturation; (ii) Investigating the enzymatic synthesis of emissive second messengers, such as c-di-NMPs, and their interactions with riboswitches and proteins (e.g., STING), (iii) Monitoring RNA?protein binding (e.g., k- turn/7LAe) and translational events, including programmed ribosomal frameshifting, which could be detrimental to native protein synthesis, but, when programmed (e.g., in viral replication) can maximize protein expression. Nucleic acids play central roles in cellular events and, as such, have immense impact on the emergence of diseases and, in turn, on human health. This necessitates the development of new effective tools for studying their recognition properties and alteration by exogenous agents. The emissive nucleoside analogs designed and prepared will be implemented in novel real time fluorescence-based assays. These investigations will further the fundamental understanding of key biological processes related to disease development and will have long-term impact on improving human health by advancing knowledge and facilitating drug discovery.